C-terminal deletion of human tissue transglutaminase enhances magnesium- dependent GTP/ATPase activity

Abstract

Tissue transglutaminase (tTG) exhibits a magnesium-dependent GTP/ATPase activity that is involved in the regulation of the cell cycle and cell receptor signaling. The portion of the molecule involved in GTP/ATP hydrolysis is unknown. We expressed and purified a series of C-terminal truncation mutants of human tTG as glutathione S-transferase fusion proteins (ΔS538, ΔE447, ΔP345, ΔC290, ΔV228, and ΔF185) to determine the effect on GTP/ATPase activity. The truncation of the C terminus did not change significantly the apparent K(m) value for either GTP or ATP. In contrast, the K(cat) value for GTP was increased by 4.6- and 3-fold for the ΔS538 and ΔE447 mutants, respectively. The ΔP345 mutant had the highest hydrolysis activity with a 34-fold increase. The hydrolysis activity then declined to 8.1-, 8.7-, and 1.9-fold for the ΔC290, ΔV228, and ΔF185 mutants, respectively. The K(cat) for ATP changed in parallel with the GTPase results. Thin layer chromatography analysis of the hydrolysis reaction products revealed that ATP was rapidly converted to ADP followed by a much slower conversion of ADP to AMP when incubated with wild type tTG or the ΔP345 mutant. There was a substantial decrease in the calcium-dependent TGase activity when the last 149 amino acid residues were deleted from the C terminus. Less than 5% of the TGase activity was detected for the ΔS538 and ΔE447 mutants. In conclusion, we have located the ATP and GTP hydrolytic domain to amino acid residues 1-185. The C terminus functions to inhibit the expression of endogenous GTP/ATPase activity of tTG, and the potential role of the C terminus in modulating this activity is discussed.